Project Summary/Abstract Nearly half a million cases of bacterial sepses are reported annually in the USA and approximately one third of the cases are fatal. Iron is a limiting nutrient in microbial growth; bacteria primarily obtain iron through production of siderophores, low molecular weight chelating agents with high ferric affinity and selectivity. The availability of iron is essential in determining the virulence of an invading pathogen. The most successful human pathogens, such as Bacillus anthracis, devise elaborate, multifaceted strategies to ensure their iron supply. This project seeks to understand siderophore transport systems: 1) from a structural level, studying the thermodynamics and kinetics of iron binding, 2) to a systemic level, following the recognition and transport of these siderophores into the bacteria, 3) to an environmental level, exploring how the surroundings, such as temperature, host immune system, presence of other bacteria and even exposure to light, affect the growth of the bacteria. We are uniquely equipped in our laboratory to carry out this range of studies and to pursue the following specific aims: 1. To understand the relationship between structure and function of siderophores. 2. To characterize siderophore-mediated iron transport in Gram-positive bacteria. 3. To explore the scope and functioning of the siderophore shuttle mechanism of microbial iron transport. 4. To further describe the mechanism of recognition of siderophores by proteins of the human immune system (siderocalin) and how the selectivity of this immune response is exploited by the most dangerous bacterial pathogens. To meet these Aims siderophore features such as thermodynamic stability and reduction potential of siderophore ferric complex, their kinetics of iron binding, and lipophilicity, will be determined for targeted siderophores and through the construction of synthetic siderophore analogs and coordination analogs we will explore siderophore function. Almost everything that is known about bacterial siderophore-mediated iron transport is in Gram-negative bacteria; Gram-positive bacteria are now our target, since this group of organisms includes many important human pathogens. We have in place collaborations to determine the crystallographic structures of membrane-associated protein receptors of Bacillus species that will complement our studies in this family. Our first report of the siderophore shuttle mechanism showed that metal exchange between two siderophores was essential for iron transport in the Gram-negative bacteria studied. We plan to see how widely distributed is this mechanism is among genera of bacteria; we now propose an extended experimental approach that incorporates the use of isotopically labeled natural siderophores. We have recently begun to develop an understanding of what we call siderophore stealth: the evasion of siderocalin binding by structural modification of the siderophore. Through the use of synthetic analogs and bacterial siderophore isolates, as well as labeled substrates and mutant proteins, we intend to describe the selectivity and physiological course of siderocalin.